Expanding use of combination antiretroviral therapy (cART) for the prevention of mother-to-child transmission (PMTCT) of HIV has dramatically decreased vertical transmission rates to <2%. The overwhelming success of PMTCT has resulted in a diminishing population of children born with perinatally acquired HIV infection on the one hand, and a mounting number of HIV-exposed uninfected (HEU) children on the other hand. Currently an estimated 20% or more of all infants born in sub-Saharan Africa are born HEU. In utero HIV/antiretroviral (ARV) and postnatal ARV treatment are known to perturb energy metabolism and could have permanent effects on the future metabolic health of HEU infants, including the development of insulin resistance. Early maladaptive changes in mitochondrial function and intermediary metabolism in HEU children exposed to ARVs may be mediated in the intrauterine environment through changes in fetal metabolic programming, and thus, may impact metabolic outcomes in these children. Through our work, we have demonstrated that exposure to specific in utero and neonatal ARVs may affect insulin sensitivity and fuel substrate utilization more than other ARVs in HEU infants early in life. Long-term and mechanistic evidence is scarce, however, in this population, particularly in sub-Saharan Africa. This study investigates the impact of in utero and postnatal HIV/ARV exposure on the metabolic health of HEU infants/children. First, we will assess whether in utero and neonatal HIV/ARV exposure is associated with changes in insulin sensitivity in HEU children from birth to 3 years of life using HIV-unexposed uninfected (HUU) infants as a comparator group. In addition, we will evaluate whether specific neonatal ARV prophylaxis regimens differ in metabolic effects amongst HEU children. Second, we will use targeted metabolomics to evaluate whether specific intermediary metabolites (short chain acylcarnitines and branched-chain amino acids) reflect or contribute to changes in insulin sensitivity in HEU and HUU children. Third, we will explore the role of the mitochondria in insulin sensitivity and intermediary metabolism amongst HEU and HUU children in an effort to understand potential mechanisms and targets for future studies. Building on our previous work with HEU and HUU infants in Africa, we propose a new prospective cohort study with a nested randomized component in HIV-infected and -uninfected woman/child dyads in Botswana. If in utero and postnatal HIV/ARV exposures are found to contribute to derangements in intermediary metabolism such that insulin sensitivity is altered early in life and HEU children are at increased risk for insulin resistance later in life, this woud impact screening and prevention strategies for diabetes in this vulnerable population and argue for further research to identify pre- and postnatal ARV regimens with superior PMTCT efficacy, but with minimal adverse metabolic consequences to the exposed fetus or infant.